Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Virtually all living organisms, including many viruses, use membranes to provide barriers against the external environment. These barriers are essential to maintain the integrity of the organism, with membrane degradation leading to cell death or viral inactivation.
Substances that disrupt membranes serve a diverse number of functions. A few of these include hygiene (soap), the immune system (many lytic pathways including the complement fixation system), and bacterial and animal toxins. These substances have diverse mechanisms; in the case of toxins, they may function by creating pores in the membrane (e.g. colicin), or by enzymatically degrading the lipids which compose the membrane itself (e.g. lipases (reviewed in Wooley and Petersen, 1994) and phospholipases (Scott et al. 1990; reviewed in Vernon and Bell 1992)).
Many lipolytic agents work indiscriminately and thus have limited therapeutic potential. In order to develop a therapeutic reagent, applicants have sought to enhance specificity. One established method for doing so, targeting, uses a molecular guide to direct the reagent specifically to its site of action.
While the idea of targeting cytotoxic agents to pathological cells is an old one (reviewed in FitzGerald and Pastan 1992, Pastan et al. 1992 and in Siegall 1994) the use of reagents which function extracellularly to disrupt membranes has not been attempted. It has several advantages.
First, such reagents would not have to cross the membrane barrier in order to be effective. Such in vivo problems as toxicity, rapid clearance, metabolic inactivation, rapid development of resistance, and low bioavailability remain major hurdles in drug development. One way to avoid or at least to lower the chance that such complications might arise is to use extracellular strategies. Although rapid clearance and development of resistance would still be problematic, extracellular therapeutics would avoid the intricate intracellular machinery, thus lowering toxicity and reducing the rate of metabolic inactivation. Moreover, they would not have to cross into the cytoplasm and thus have higher bioavailability.
Second, such reagents would be effective against the virions of enveloped viruses (such as herpes, influenza, or retroviruses such as HIV), which are resistant to conventional directed toxins. Indeed viruses, which lack cellular biosynthetic repair mechanisms, would be uniquely susceptible to membrane degradation.
As an initial trial, applicants have sought to combine specificity with anti-viral potency through the creation of targeted phospholipases. While applicants have focused on phospholipases as an initial test case, it may be that a different application of the general idea, to specifically target lipolytic agents against pathological cells and enveloped virions, will ultimately prove to be more useful medically.